[SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA'2B, but not UmuD'2c (PolV) polymerase].

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength > 320 nm are increased whenthe wild-type host (Escherichia coli uvr+) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as "W reactivation" and "W mutagenesis." It is shown that 8-MOP monoadducts in λ DNA in- duce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA B, but not PolV (UmuD'2C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc+ in E. coli uvr-.

In vitro photo-induced cytotoxic activity of Citrus bergamia and C. medica L. cv. Diamante peel essential oils and identified active coumarins.

CONTEXT: The search for innovative therapeutic approaches is gaining more interest in clinical oncology. OBJECTIVE: In the present investigation we reported the chemical profile and the photo-induced cytotoxic activity of two endemic Calabrian Citrus species (Rutaceae): Citrus bergamia Risso & Poit. and Citrus medica L. cv. Diamante. MATERIALS AND METHODS: Essential oils were obtained by hydrodistillation and analyzed by GC and GC/MS. In order to evaluate the cytotoxic activity two melanoma models, such as amelanotic melanoma C32 and malignant melanoma A375, were used. RESULTS: The essential oil of C. bergamia was characterized by limonene, linalyl acetate, gamma-terpinene, linalool and beta-pinene as major components. The most abundant compounds of C. medica cv. Diamante oil were limonene, gamma-terpinene, citral, geranial, beta-pinene and alpha-pinene. Two coumarins, bergapten and citropten, were also identified in C. bergamia and C. medica cv. Diamante, respectively and tested for biological activity. Both C. bergamia and C. medica cv. Diamante oils exhibited a selective interesting activity against the A375 cell line with IC(50) values of 79.3 and 89.1 microg/mL, respectively, after 100 min exposure to UV irradiation. The strong antiproliferative activity demonstrated with bergapten (IC(50) value of 71.3 microg/mL after 20 min of irradiation) was not found with citropten. DISCUSSION AND CONCLUSION: Our study suggested that UV irradiation is effective in activating essential oils and in particular bergapten. This phototoxicity may be considered as a treatment option in some cases of lentigo maligna or lentigo maligna melanoma.

Erythroid induction of chronic myelogenous leukemia K562 cells following treatment with a photoproduct derived from the UV-A irradiation of 5-methoxypsoralen.

Induction of terminal erythroid differentiation can be an efficient strategy to inhibit proliferation of chronic myelogenous leukemia cells. Psoralens, well-known photo-chemotherapeutic agents, were found to be efficient at inducing erythroid differentiation of K562 cells, an in vitro cell line isolated from the pleural effusion of a patient with chronic myelogenous leukemia in blast crisis. The effects of crude pre-irradiated solutions of 5-methoxypsoralen on erythroid differentiation of human leukemic K-562 cells were evaluated. The major photoproduct was characterized and analyzed, and it was found to induce erythroid differentiation of K562 cells and inhibit NF-kappaB/DNA interactions.

Furocoumarins photolysis products induce differentiation of human erythroid cells.

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells.

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.

Spectroscopic detection of photoproducts in lecithin model system after 8-methoxypsoralen plus UV-A treatment.

Photoreactions of 8-methoxypsoralen (8-MOP) in the presence of synthetic lecithins esterified at the beta-position with linoleic/oleic and gamma-palmitic acid (PCd2/d1pal) have been studied. Following UV-A (320-400 nm) irradiation, the photoproducts separated by thin-layer chromatography are analysed by UV absorption spectroscopy, nuclear magnetic resonance and mass spectroscopy. The new isolated products are lecithin double cyclobutane adducts, PC-(8-MOP)2, fatty acid-8-MOP split adducts from phosphatidylcholine and lecithin adducts with photo-oxidized 8-MOP. The photolysis performed in the presence of 8-MOP is related to the previously reported lecithin cyclobutane adducts with psoralen. A hypothetical scheme of lecithin photolysis under PUVA (psoralen + UV-A) treatment is proposed. We suggest that photolysis of lecithin may have a significant role in the chain of reactions triggered in cell membrane submitted to PUVA treatment.

5-Methoxypsoralen photoadduct formation: conversion of monoadducts to crosslinks.

5-Methoxypsoralen is often substituted for 8-methoxypsoralen in the photochemotherapy of psoriasis even though the nature of the resulting photadducts in cellular DNA has not been determined. A recent molecular mechanics study with a model oligonucleotide predicted that intercalated 5-methoxypsoralen molecules would tend to favor the preferential formation of 3,4-monoadducts. Such a result would be contrary to the photoadduct patterns observed with other psoralens. In this study we show that 5-methoxypsoralen photoadducts formation is, in fact, very similar to that for other psoralens, i.e., the primary photoadduct is the 4',5'-monoadduct which can be quantitatively converted to crosslink.

Treatment with 8-MOP and UVA enhances MHC class I synthesis in RMA cells: preliminary results.

The response of psoriasis and cutaneous T-cell lymphoma to treatment with 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet light (UVA) is only partly understood. Psoralens form photoadducts within the DNA after activation by UVA and this damage leads to the inhibition of DNA synthesis. Additionally, it has been shown that different forms of DNA damage can induce a stress response, leading to upregulation of selected products. Among these are the major histocompatibility complex (MHC) class I genes. Thus the aim of the present study was to assess the rate of synthesis of MHC class I proteins in murine T-cell lymphoma cells (RMA) after treatment with 8-MOP and UVA. RMA cells were treated with 8-MOP (50-200 ng ml-1) and UVA (1 J.cm-2) and metabolically labelled with 35S-methionine 4 and 24 h after treatment. MHC class I synthesis was determined by immunoprecipitation of the cell lysates with an anti-Kb monoclonal antibody, Y3. After 4 h, treated and untreated cells demonstrated no differences in the rate of MHC class I synthesis. However, after 24 h a dose-dependent increase in MHC class I synthesis was observed. This increase in MHC class I expression could be responsible, at least partly, for the responses observed in patients treated with photopheresis.

Photoinduction of micronuclei by 4,4',6-trimethylangelicin and 8-methoxypsoralen in different experimental models.

The frequencies of micronuclei induced by treatment with 4,4',6-trimethylangelicin (TMA) and 8-methoxypsoralen (8-MOP) have been compared in the following experimental models: (1) peripheral normochromatic erythrocytes (NCE) during 10 days after single p.o. administration of TMA or 8-MOP in male and female mice; (2) peripheral NCE during photocarcinogenesis by TMA or 8-MOP topically administered to female mice; (3) primary cultures of human skin fibroblasts treated with TMA or 8-MOP. The frequency of micronuclei in peripheral NCE of mice (both sexes) was significantly enhanced after p.o. administration of TMA or 8-MOP. This latter was more active than TMA in inducing chromosomal damage. No increased frequencies of micronuclei in peripheral NCE were detected in mice subjected to TMA or 8-MOP photocarcinogenic treatment, even when malignancies developed. In human fibroblast cultures, at equimolar concentrations, the induction of lethal effects by TMA in the presence of 365-nm radiation was higher than that exerted by 8-MOP. At equal survival, however, TMA showed practically the same activity as 8-MOP in the induction of micronuclei. Our findings provide evidence of genotoxicity by TMA administered p.o. without irradiation and give further information about photogenotoxicity of these substances.

Photoreaction of 5-methoxypsoralen with thymidine. Isolation and characterization of a pyrone-side monoadduct involving the pyrimidine methyl group.

The UVA-mediated photoreaction of 5-methoxypsoralen (5-MOP) with thymidine has been investigated in the dry state. Under these conditions, the main products are 5-MOP pyrone-side monoadducts to thymidine. We report the isolation and characterization of an unusual 5-MOP-thymidine photoproduct. The assignment of the photoadduct was achieved on the basis of extensive spectroscopic measurements (UV, mass spectrometry (MS), 1H and 13C nuclear magnetic resonance (NMR), nuclear Overhauser effect (NOE) experiments). The formation of the photoadduct, which is rationalized in terms of a radical mechanism, appears to involve, in a covalent bond, the C-4 pyrone moiety of 5-MOP and the methyl group of thymidine.

The excitation of 8-methoxypsoralen with visible light: reversed phase HPLC quantitation of monoadducts and cross-links.

The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA, 320-400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1,000 ng/mL) and 419 nm light (up to 12 J/cm2). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.

Use of 8-methoxypsoralen and long wavelength ultraviolet radiation for decontamination of platelet concentrates.

Transmission of viral diseases through blood products remains a problem in transfusion medicine. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP). This system is capable of inactivating 25-30 logs/hour of bacteria E. coli or S. aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC. Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent. After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hours. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

Studies on 8-methoxypsoralen tolbutamide interactions. 2. Studies after U.V.-A irradiation in vitro and in vivo.

Previous studies had evidenced that tolbutamide has influence on the complex formation between 8-methoxypsoralen (8-MOP) and serum albumin, both in vitro and in vivo, partially displacing it from its binding site. We have now extended this research by studying competition under UV-A irradiation, thus approaching the conditions which occur during PUVA therapy more closely. Research was first carried out in vitro, studying tolbutamide competing with bound 8-MOP in human serum after irradiation with UV-A. From the data obtained, it appears that the displacement is not influenced to any significant extent by irradiation. Experiments were then carried out in vivo on mice treated with the two drugs and irradiated with UV-A in conditions analogous to those generally used for PUVA therapy; the amount of 8-MOP localized in blood serum and in various organs was then determined. Concerning distribution of 8-MOP, results confirmed what had been previously obtained; concerning irradiation, results showed that the effect on the 8-MOP displacement by tolbutamide was not statistically significative in the most organs considered. A slight difference in respect to the results obtained without irradiation was observed only in the cases of skin, eyes and ears.

Activation of hyaluronidase by 8-methoxypsoralen-polyamine photoproducts.

Ultraviolet radiation of 8-methoxypsoralen (8-MOP) in the presence of various polyamines resulted in stable photoproducts that were very soluble in water and showed hyaluronidase-activating properties. Among them, the photoproducts obtained from the reaction systems of 8-MOP-spermine and 8-MOP-spermidine markedly activated hyaluronidase. The enzyme activity was not affected by 8-MOP alone and the photoproduct of 8-MOP (8-MOP-P). From these facts, it was suggested that the photoproducts with hyaluronidase-activating properties might play an important role in the onset of 8-MOP-induced photosensitivity.

Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light--I. Assay development.

Two complementary assay systems have been adapted for the detection of compounds which may form mutagenic photoproducts during exposure to UV light from an Osram Ultra-Vitalux sunlamp as used in the evaluation of the effectiveness of sun filters. The effects of UVA and of UVB were evaluated. A bacterial plate test using Escherichia coli strain WP2 allowed the bacteria, co-plated with test chemical in soft agar, to be irradiated with various doses of UV light. Mutagenesis was assessed by scoring numbers of tryptophan-independent colonies. The chosen reference compound was 8-methoxypsoralen (8-MOP) which was non-mutagenic alone at the highest dose tested (1000 micrograms/plate). Following simultaneous exposure of bacteria to 8-MOP and doses of UV light which alone had little effect, large numbers of revertants were scored. Numbers of mutants were dependent upon the doses of both 8-MOP and of UV light. The second test system involved the exposure of Chinese hamster ovary cells to UV light in the presence of test chemical to determine the clastogenic effects of photoproducts. Treatment with 8-MOP alone up to 50 micrograms/ml was not clastogenic but concomitant exposure to non-damaging doses of UV light caused large increases in the incidence of chromosome aberrations of all types. Damage was again dependent on the doses of both components. Two additional photoactive compounds, para-aminobenzoic acid and chlorpromazine both show photoclastogenic but not photomutagenic properties. These two complementary assay systems take advantage of using no-effect levels of UV light as a baseline against which photomutagenicity readily can be compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation and removal of 8-MOP-DNA photoadducts in keratinocytes: effects of calcium concentration and retinoids.

8-methoxypsoralen (8-MOP)-DNA photoadducts were quantified in freshly isolated human and murine keratinocytes and cultured keratinocyte cell lines after in vitro treatment with 8-MOP (1-200 ng/ml) and ultraviolet A (UVA; 0.2-24.0 J/cm2). Greater doses of 8-MOP and UVA led to proportionately greater numbers of photoadducts, with a dose reciprocity relationship between the amounts of 8-MOP and UVA. No significant difference in photoadduct formation was observed between basal and differentiated cells. However, the transformed keratinocyte cell lines showed fewer photoadducts than did normal keratinocytes, which appeared to be correlated with the finding that the adduct formation was inhibited in normal keratinocytes cultured with phorbol 12-myristate 13-acetate, because this agent leads to epidermal hyperproliferation. In viable keratinocytes that were treated with a sublethal dose of 8-MOP and UVA (15 ng/ml and 1 J/cm2, respectively), 54% of photoadducts formed were removed over a 20-h period. Adduct removal depended on the calcium concentration in the media; cells cultured in standard high calcium levels showed a higher removal rate than those cultured in low-calcium media. The addition of retinoids (etretinate, acitretin, and 13-cis retinoic acid) to the culture induced 55 to 80% of suppression of the adduct removal. The calcium ionophore A23187 partially restored the suppression of photoadduct removal induced by retinoids. The present studies suggest that calcium performs an important role in the photoadduct removal and raise the possibility that the synergism of systemic retinoids and psoralen plus UVA photochemotherapy relates to the former's inhibition of repair of 8-MOP photoadducts in DNA.

Genotoxicity of bergapten and bergamot oil in Saccharomyces cerevisiae.

In order to determine the genotoxic potential of bergapten (5-methoxypsoralen (5-MOP] and bergamot oil (BO), the genetic effects of 5-MOP and BO (containing equivalent amounts of 5-MOP) were studied in haploid and diploid yeast (Saccharomyces cerevisiae) using solar simulated radiation (SSR). At equal doses of SSR, equal concentrations of 5-MOP alone or 5-MOP in BO have a similar influence on survival and on the induction of cytoplasmic "petite" mutations, reverse and forward mutations, mitotic gene conversion and genetically aberrant colonies including mitotic crossing over. No reciprocity is found between SSR dose and 5-MOP concentration for cytotoxic, mutagenic and recombinogenic effects. In the presence of chemical filters (Parsol 1789, a UVA filter, and Parsol MCX, a cinnamate derivative acting as a UVB filter) considerable protection is observed against the induction of genetic effects by 5-MOP and BO containing 5-MOP in haploid and diploid cells. As indicated by the lower induction kinetics, the protection is higher than expected from the light-absorbing properties, suggesting photochemical interaction. The protection is slightly higher for BO than for 5-MOP. The induction of genetic effects by 5-MOP alone or BO containing 5-MOP is independent of oxygen. Experiments on suction blister fluids taken from patients after topical treatment with BO containing 5-MOP indicate that in comparison with water the bioavailability and thus the genotoxic effects of the compounds are decreased. Moreover, in addition to the filtering effect against the photoinduced genotoxic effects of BO, the presence of chemical filters apparently reduces the penetration of BO containing 5-MOP and provides a reduction in biological effectiveness.

Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: evaluation and modification of risk of human use in an albino mouse skin model.

The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.